Overexpression of ClC-3 Chloride Channel in Chondrosarcoma: An In Vivo Immunohistochemical Study with Tissue Microarray

We have previously shown [B. Illek, H. Fischer, G. F. Santos, J. H. Widdicombe, T. E. Machen, and W. W. Reenstra, Am. J. Physiol. 268 (Cell Physiol. 37): C886-C893, 1995] that genistein, a tyrosine kinase inhibitor, activates the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel in NIH/3T3 cells that have been stably transfected with an expression vector for the CFTR (NIH-CFTR cells). In this study, we present evidence suggesting that both genistein and the serine/threonine protein phosphatase (PPase) inhibitor calyculin A activate the CFTR by inhibiting PPase activity. As measured by 125I efflux, genistein and calyculin A stimulate the CFTR to approximately 50% of the maximal activity with forskolin. Neither agonist increases CFTR activity at saturating forskolin concentrations, but genistein and calyculin A have an additive effect on CFTR activity. Forskolin, but neither genistein nor calyculin A, stimulates protein kinase A(PKA) activity. The PKA inhibitor H-89 inhibits CFTR activation and in vivo phosphorylation by all three agonists. Proteolytic digestion of in vivo phosphorylated CFTR suggests that the CFTR is phosphorylated on the same sites during stimulation with genistein and forskolin but on different sites stimulation with calyculin A. The data suggest that genistein and calyculin A inhibit different PPase activities, allowing CFTR phosphorylation and partial stimulation, by a basal PKA activity.

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Dynamic measurement of cytosolic pH and [NO3−] uncovers the role of the vacuolar transporter AtCLCa in cytosolic pH homeostasis

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2007580117 ◽

2020 ◽

Vol 117 (26) ◽

pp. 15343-15353 ◽

Cited By ~ 2

Author(s):

Elsa Demes ◽

Laetitia Besse ◽

Paloma Cubero-Font ◽

Béatrice Satiat-Jeunemaitre ◽

Sébastien Thomine ◽

...

Keyword(s):

Chloride Channel ◽

Loss Of Function ◽

Ion Transporters ◽

Ph Homeostasis ◽

Cytosolic Ph ◽

Cellular Processes ◽

Plasma Membrane Proton Pump ◽

Cytosolic Acidification

Ion transporters are key players of cellular processes. The mechanistic properties of ion transporters have been well elucidated by biophysical methods. Meanwhile, the understanding of their exact functions in cellular homeostasis is limited by the difficulty of monitoring their activity in vivo. The development of biosensors to track subtle changes in intracellular parameters provides invaluable tools to tackle this challenging issue. AtCLCa (Arabidopsis thalianaChloride Channel a) is a vacuolar NO3−/H+exchanger regulating stomata aperture inA.thaliana. Here, we used a genetically encoded biosensor, ClopHensor, reporting the dynamics of cytosolic anion concentration and pH to monitor the activity of AtCLCa in vivo inArabidopsisguard cells. We first found that ClopHensor is not only a Cl−but also, an NO3−sensor. We were then able to quantify the variations of NO3−and pH in the cytosol. Our data showed that AtCLCa activity modifies cytosolic pH and NO3−. In an AtCLCa loss of function mutant, the cytosolic acidification triggered by extracellular NO3−and the recovery of pH upon treatment with fusicoccin (a fungal toxin that activates the plasma membrane proton pump) are impaired, demonstrating that the transport activity of this vacuolar exchanger has a profound impact on cytosolic homeostasis. This opens a perspective on the function of intracellular transporters of the Chloride Channel (CLC) family in eukaryotes: not only controlling the intraorganelle lumen but also, actively modifying cytosolic conditions.

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